This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The endocytic pathway functions to segregate ingested macromolecules that are destined to be degraded in the lysosome from molecules that are either recycled back to the plasma membrane or routed toward other intracellular destinations. One of the most recognizable endocytic compartments is the multivesicular body (MVB), which contains within its lumen membrane-enclosed vesicles that are formed by inward budding of the limiting endosomal membrane. Many activated cell-surface receptors that control cell proliferation are down-regulated through endocytosis and sorting into the lumenal vesicles of MVBs. These vesicles and their contents are subsequently degraded by lysosomal hydrolases upon fusion of the limiting MVB membrane with the lysosome. Preliminary tomograms have shown that wild-type budding yeasts contain MVBs that are similar to those in higher eukaryotic cells. A novel genetic screen was used to uncover the BRO1 gene, which encodes a conserved, soluble cytoplasmic protein that associates with endosomal membranes. The Bro1 protein may be recruited from the cytoplasm to the endosome in order to coordinate inward invagination of the endosomal membrane and budding of vesicles toward the compartment's lumen. To test this hypothesis, the function and localization of Bro1 proteins that have domain-specific mutations will be analyzed. Initially, we completed 9 dual-axis tomograms of BRO1-deleted cells and found that these cells contained aberrant membrane compartments with three distinct morphologies. These included stacked cisternae, stacked cisternae that were curved into a C-shape, and curved cisternae enclosing a spherical compartment. Future studies will use genetic and biochemical analysis to identify proteins that cooperate with the wild-type Bro1 protein in MVB vesicle formation. The Odorizzi lab has recently hired a full time technician, Matthew West who is using the Resource facilities to work on additional strains that have mutations in other genes required for MVB sorting. Together, these studies should define the functional role of the Bro1 protein in MVB vesicle formation and identify new yeast proteins with human homologues that could serve as targets for therapeutics in human diseases.